Polynucleotides and polypeptides BASB033 from neisseria meningitidis and their uses

ABSTRACT

The invention provides BASB033 polypeptides and polynucleotides encoding BASB033 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.

FIELD OF THE INVENTION

This invention relates to polynucleotides, (herein referred to as“BASB033 polynucleotide(s)”), polypeptides encoded by them (referred toherein as “BASB033” or “BASB033 polypeptide(s)”), recombinant materialsand methods for their production. In another aspect, the inventionrelates to methods for using such polypeptides and polynucleotides,including vaccines against bacterial infections. In a further aspect,the invention relates to diagnostic assays for detecting infection ofcertain pathogens.

BACKGROUND OF THE INVENTION

Neisseria meningitidis (meningococcus) is a Gram-negative bacteriumfrequently isolated from the human upper respiratory tract. Itoccasionally causes invasive bacterial diseases such as bacteremia andmeningitis. The incidence of meningococcal disease shows geographicalseasonal and annual differences (Schwartz, B., Moore, P. S., Broome, C.V.; Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Most diseasein temperate countries is due to strains of serogroup B and varies inincidence from 1-10/100,000/year total population sometimes reachinghigher values (Kaczmarski, E. B. (1997), Commun. Dis. Rep. Rev. 7:R55-9, 1995; Scholten, R. J. P. M., Bijlmer, H. A., Poolman, J. T. etal. Clin. Infect. Dis. 16: 237-246, 1993; Cruz, C., Pavez, G., Aguilar,E., et al. Epidemiol. Infect. 105:119-126, 1990).

Epidemics dominated by serogroup A meningococci, mostly in centralAfrica, are encountered, sometimes reaching levels up to1000/100,000/year (Schwartz. B., Moore. P. S., Broome. C. V. Clin.Microbiol. Rev. 2 (Supplement). S18-S24, 1989). Nearly all cases as awhole of meningococcal disease are caused by serogroup A, B, C, W-135and Y meningococci and a tetravalent A, C, W-135, Y polysaccharidevaccine is available (Armand, J., Arminjon, F., Mynard, M. C., Lafaix,C., J. Biol. Stand. 10: 335-339, 1982).

The polysaccharide vaccines are currently being improved by way ofchemical conjugating them to carrier proteins (Lieberman, J. M., Chiu,S. S., Wong. V. K., et al. JAMA 275: 1499-1503, 1996).

A serogroup B vaccine is not available, since the B capsularpolysaccharide was found to be nonimmunogenic, most likely because itshares structural similarity to host components (Wyle, F. A.,Artenstein, M. S., Brandt, M. L. et al. J. Infect. Dis. 126: 514-522,1972; Finne, J. M., Leinonen, M., Mäkelä, P. M. Lancet ii.: 355-357,1983).

For many years efforts have been initiated and carried out to developmeningococcal outer membrane based vaccines (de Moraes, J. C., Perkins,B., Camargo, M. C. et al. Lancet 340: 1074-1078, 1992; Bjune, G., Hoiby,E. A. Gronnesby, J. K. et al. 338: 1093-1096, 1991). Such vaccines havedemonstrated efficacies from 57%-85% in older children (>4 years) andadolescents.

Many bacterial outer membrane components are present in these vaccines,such as PorA. PorB, Rmp, Opc, Opa, FrpB and the contribution of thesecomponents to the observed protection still needs futher definition.Other bacterial outer membrane components have been defined by usinganimal or human antibodies to be potentially relevant to the inductionof protective immunity, such as TbpB and NspA (Martin, D., Cadieux, N.,Hamel, J., Brodeux, B. R., J. Exp. Med. 185: 1173-1183, 1997; Lissolo,L., Maître-Wilmotte, C., Dumas, p. et al., Inf. Immun. 63: 884-890,1995). The mechanisms of protective immunity will involve antibodymediated bactericidal activity and opsonophagocytosis.

A bacteremia animal model has been used to combine all antibody mediatedmechanisms (Saukkonen, K., Leinonen, M., Abdillahi, H. Poolman, J. T.Vaccine 7: 325-328, 1989). It is generally accepted that the latecomplement component mediated bactericidal mechanism is crucial forimmunity against meningococcal disease (Ross, S. C., Rosenthal P. J.,Berberic, H. M., Densen, P. J. Infect. Dis. 155: 1266-1275, 1987).

The frequency of Neisseria meningitidis infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Neisseria meningitidis strains that are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

SUMMARY OF THE INVENTION

The present invention relates to BASB033, in particular BASB033polypeptides and BASB033 polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingprevention and treatment of microbial diseases, amongst others. In afurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with microbial infections and conditions associatedwith such infections, such as assays for detecting expression oractivity of BASB033 polynucleotides or polypeptides.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to BASB033 polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of BASB033 of Neisseriameningitidis, which is related by amino acid sequence homology toKlebsiella pneumoniae outer membrane phospholipase A protein. Theinvention relates especially to BASB033 having the nucleotide and aminoacid sequences set out in SEQ ID NO:1,3 and SEQ ID NO:2,4 respectively.It is understood that sequences recited in the Sequence Listing below as“DNA” represent an exemplification of one embodiment of the invention,since those of ordinary skill will recognize that such sequences can beusefully employed in polynucleotides in general, includingribopolynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of the BASB033 polynucleotide sequences withidentity to SEQ ID NO:1 indicated by a dot and missing nucleotidesindicated by a dash.

FIG. 2 is an alignment of the BASB033 polypeptide sequences withidentity to SEQ ID NO:2 indicated by a dot and missing amino acidsindicated by a dash.

FIG. 3 is a photograph of an SDS-PAGE gel electrophoresis showingexpression and purification of recombinant BASB033 in E. coli.

FIG. 4 is a photograph of an SDS-PAGE gel electrophoresis showingexpression and purification of recombinant BASB033 in E. coli.

FIG. 5 is a graph of Anti-BASB033 antibodies by the ELISA method.

FIG. 6 shows specific BASB033 antibodies by Western Blot.

FIG. 7 shows recognition of the BASB033 protein on several NmB strainswith BASB033 immunized mice sera.

FIG. 8 shows recognition of the BASB033 protein on several NmB strainswith BASB033 immunized mice sera.

FIG. 9 shows anti-BASB033 antibodies in convalescent sera (part B) andin Immunized mice (part A).

POLYPEPTIDES

In one aspect of the invention there are provided polypeptides ofNeisseria meningitidis referred to herein as “BASB033” and “BASB033polypeptides” as well as biologically. diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

The present invention further provides for:

(a) an isolated polypeptide which comprises an amino acid sequence whichhas at least 85% identity, more preferably at least 90% identity, yetmore preferably at least 95% identity, most preferably at least 97-99%or exact identity, to that of SEQ ID NO:2, 4;

(b) a polypeptide encoded by an isolated polynucleotide comprising apolynucleotide sequence which has at least 85% identity, more preferablyat least 90% identity, yet more preferably at least 95% identity, evenmore preferably at least 97-99% or exact identity to SEQ ID NO:1, 3 overthe entire length of SEQ ID NO:1, 3 respectively; or

(c) a polypeptide encoded by an isolated polynucleotide comprising apolynucleotide sequence encoding a polypeptide which has at least 85%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity, to the amino acid sequence of SEQ ID NO:2, 4;

The BASB033 polypeptides provided in SEQ ID NO:2,4 are the BASB033polypeptides from Neisseria meningitidis strains ATCC13090 and H44/76.

The invention also provides an immunogenic fragment of a BASB033polypeptide, that is, a contiguous portion of the BASB033 polypeptidewhich has the same or substantially the same immunogenic activity as thepolypeptide comprising the amino acid sequence of SEQ ID NO:2,4. That isto say, the fragment (if necessary when coupled to a carrier) is capableof raising an immune response which recognises the BASB033 polypeptide.Such an immunogenic fragment may include, for example, the BASB033polypeptide lacking an N-terminal leader sequence, and/or atransmembrane domain and/or a C-terminal anchor domain. In a preferredaspect the immunogenic fragment of BASB033 according to the inventioncomprises substantially all of the extracellular domain of a polypeptidewhich has at least 85% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:2,4 over the entire length of SEQID NO:2

A fragment is a polypeptide having an amino acid sequence that isentirely the same as part but not all of any amino acid sequence of anypolypeptide of the invention. As with BASB033 polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of SEQ ID NO:2,4 or of variantsthereof, such as a continuous series of residues that includes an amino-and/or carboxyl-terminal amino acid sequence. Degradation forms of thepolypeptides of the invention produced by or in a host cell, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, colt and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2,4, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.4.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis: therefore, these fragments may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination.

The polypeptides, or immunogenic fragments, of the invention may be inthe form of the “mature” protein or may be a part of a larger proteinsuch as a precursor or a fusion protein. It is often advantageous toinclude an additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purificationsuch as multiple histidine residues, or an additional sequence forstability during recombinant production. Furthermore, addition ofexogenous polypeptide or lipid tail or polynucleotide sequences toincrease the immunogenic potential of the final molecule is alsoconsidered.

In one aspect, the invention relates to genetically engineered solublefusion proteins comprising a polypeptide of the present invention, or afragment thereof, and various portions of the constant regions of heavyor light chains of immunoglobulins of various subclasses (IgG, IgM, IgA,IgE). Preferred as an immunoglobulin is the constant part of the heavychain of human IgG, particularly IgG1, where fusion takes place at thehinge region. In a particular embodiment, the Fc part can be removedsimply by incorporation of a cleavage sequence which can be cleaved withblood clotting factor Xa.

Furthermore, this invention relates to processes for the preparation ofthese fusion proteins by genetic engineering, and to the use thereof fordrug screening, diagnosis and therapy. A further aspect of the inventionalso relates to polynucleotides encoding such fusion proteins. Examplesof fusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

The proteins may be chemically conjugated, or expressed as recombinantfusion proteins allowing increased levels to be produced in anexpression system as compared to non-fused protein. The fusion partnermay assist in providing T helper epitopes (immunological fusionpartner), preferably T helper epitopes recognised by humans, or assistin expressing the protein (expression enhancer) at higher yields thanthe native recombinant protein. Preferably the fusion partner will beboth an immunological fusion partner and expression enhancing partner.

Fusion partners include protein D from Haemophilus influenzae and thenon-structural protein from influenzae virus, NS1 (hemaigglutinin).Another fusion partner is the protein known as LytA. Preferably the Cterminal portion of the molecule is used. LytA is derived fromStreptococcuts pneumoniae which synthesize an N-acetyl-L-alanineamidase, amidase LytA, (coded by the lytA gene {Gene, 43 (1986) pare265-272}) an autolysin that specifically degrades certain bonds in thepeptidoglycan backbone. The C-terminal domain of the LytA protein isresponsible for the affinity to the choline or to some cholineanalolgues such as DEAE. This property has been exploited for thedevelopment of E.coli C-LytA expressing plasmids useful for expressionof fusion proteins. Purification of hybrid proteins containing theC-LytA fragment at its amino terminus has been described {Biotechnology:10, (1992) page 795-798}. It is possible to use the repeat portion ofthe LytA molecule found in the C terminal end starting at residue 178,for example residues 188-305.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions. whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

It is most preferred that a polypeptide of the invention is derived fromNeisseria meningitidis, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodeBASB033 polypeptides, particularly polynucleotides that encode thepolypeptide herein designated BASB033.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding BASB033 polypeptidescomprising a sequence set out in SEQ ID NO:1,3 which includes a fulllength gene, or a variant thereof.

The BASB033 polynucleotides provided in SEQ ID NO:1,3 are the BASB033polynucleotides from Neisseria meningitidis strains ATCC 13090 andH44/76.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing BASB033 polypeptides andpolynucleotides, particularly Neisseria meningitidis BASB033polypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B-and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a BASB033polypeptide having a deduced amino acid sequence of SEQ ID NO:2,4 andpolynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is aBASB033 polypeptide from Neisseria meningitidis comprising or consistingof an amino acid sequence of SEQ ID NO:2,4 or a variant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in SEQ ID NO:1, 3 a polynucleotide of the invention encodingBASB033 polypeptide may be obtained using standard cloning and screeningmethods, such as those for cloning, and sequencing chromosomal DNAfragments from bacteria using Neisseria meningitidis cells as startingmaterial, followed by obtaining a full length clone. For example, toobtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in SEQ ID NO:1,3, typically a library ofclones of chromosomal DNA of Neisseria meningitidis in E. coli or someother suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent hybridization conditions. By sequencing the individualclones thus identified by hybridization with sequencing primers designedfrom the original polypeptide or polynucleotide sequence it is thenpossible to extend the polynucleotide sequence in both directions todetermine a full length gene sequence. Conveniently, such sequencing isperformed, for example, using denatured double stranded DNA preparedfrom a plasmid clone. Suitable techniques are described by Maniatis, T.,Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90and Sequencing Denatured Double-Stranded DNA Templates 13.70). Directgenomic DNA sequencing may also be performed to obtain a full lengthgene sequence. Illustrative of the invention, each polynucleotide setout in SEQ ID NO:1,3 was discovered in a DNA library derived fromNeisseria meningitidis.

Moreover, each DNA sequence set out in SEQ ID NO:1,3 contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in SEQ ID NO:2, 4 with a deduced molecular weightthat can be calculated using amino acid residue molecular weight valueswell known to those skilled in the art.

The polynucleotide of SEQ ID NO:1, between the start codon at nucleotidenumber 1 and the stop codon which begins at nucleotide number 1126 ofSEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.

The polynucleotide of SEQ ID NO:3, between the start codon at nucleotidenumber 1 and the stop codon which begins at nucleotide number 1123 ofSEQ ID NO:3, encodes the polypeptide of SEQ ID NO:4.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of:

(a) a polynucleotide sequence which has at least 85% identity, morepreferably at least 90% identity, yet more preferably at least 95%identity, even more preferably at least 97-99% or exact identity to SEQID NO:1,3 over the entire length of SEQ ID NO:1,3 respectively; or

(b) a polynucleotide sequence encoding a polypeptide which has at least85% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, even more preferably at least 97-99% or 100%exact, to the amino acid sequence of SEQ ID NO:2, 4 over the entirelength of SEQ ID NO:2, 4 respectively.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Neisseriameningitidis, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions (for example, using a temperature in the range of 45-65° C.and an SDS concentration from 0.1-1%) with a labeled or detectable probeconsisting of or comprising the sequence of SEQ ID NO:1,3 or a fragmentthereof; and isolating a full-length gene and/or genomic clonescontaining said polynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in SEQ ID NO: 1,3. Also provided by the invention is a coding sequence for a maturepolypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites. Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals.

The polynucleotide sequence may also comprise additional coding sequenceencoding, additional amino acids. For example, a marker sequence thatfacilitates purification of the fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

The nucleotide sequence encoding BASB033 polypeptide of SEQ ID NO:2, 4may be identical to the polypeptide encoding sequence contained innucleotides 1 to 1125 of SEQ ID NO:1, or the polypeptide encodingsequence contained in nucleotides 1 to 1122 of SEQ ID NO:3,respectively. Alternatively it may be a sequence, which as a result ofthe redundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO:2, 4.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Neisseria meningitidis BASB033having an amino acid sequence set out in SEQ ID NO:2, 4. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA recognization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of SEQ ID NO:2, 4.

Fragments of polynucleotides of the invention may be used, for example,to synthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingBASB033 variants, that have the amino acid sequence of BASB033polypeptide of SEQ ID NO:2, 4 in which several, a few, 5 to 10, 1 to 5,1 to 3, 2, 1 or no amino acid residues are substituted, modified,deleted and/or added, in any combination. Especially preferred amongthese are silent substitutions, additions and deletions, that do notalter the properties and activities of BASB033 polypeptide.

Further preferred embodiments of the invention are polynucleotides thatare at least 85% identical over their entire length to a polynucleotideencoding BASB033 polypeptide having an amino acid sequence set out inSEQ ID NO:2, 4, and polynucleotides that are complementary to suchpolynucleotides. In this regard, polynucleotides at least 90% identicalover their entire length to the same are particularly preferred, andamong these particularly preferred polynucleotides, those with at least95% are especially preferred. Furthermore, those with at least 97% arehighly preferred among those with at least 95%, and among these thosewith at least 98% and at least 99% are particularly highly preferred,with at least 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of SEQ ID NO:1, 3.

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to BASB033 polynucleotide sequences, such as thosepolynucleotides in SEQ ID NO:1, 3.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6). 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1, 3 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO:1, 3, or a fragment thereof; and isolating said polynucleotidesequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding BASB033 and to isolatecDNA and genomic clones of other genes that have a high identity,particularly high sequence identity, to the BASB033 gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have less than 30 nucleotide residues or basepairs.

A coding region of a BASB033 gene may be isolated by screening using aDNA sequence provided in SEQ ID NO:1, 3 to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the invention is then used to screen a library of cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al. PNAS USA 85: 8998-9002, 1988). Recentmodifications of the technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.) for example, have significantly simplifiedthe search for longer cDNAs. In the Marathon™ technology, cDNAs havebeen prepared from mRNA extracted from a chosen tissue and an ‘adaptor’sequence ligated onto each end. Nucleic acid amplification (PCR) is thencarried out to amplify the “missing” 5′ end of the DNA using acombination of gene specific and adaptor specific oliconucleotideprimers. The PCR reaction is then repeated using “nested” primers, thatis, primers designed to anneal within the amplified product (typicallyan adaptor specific primer that anneals further 3′ in the adaptorsequence and a gene specific primer that anneals further 5′ in theselected gene sequence). The products of this reaction can then beanalyzed by DNA sequencing and a full-length DNA constructed either byjoining the product directly to the existing DNA to give a completesequence, or carrying, out a separate full-length PCR using the newsequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of SEQ ID NOS:1-4 may be used in the processes herein asdescribed, but preferably for PCR, to determine whether or not thepolynucleotides identified herein in whole or in part are transcribed inbacteria in infected tissue. It is recognized that such sequences willalso have utility in diagnosis of the stage of infection and type ofinfection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is the mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from the mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

In accordance with an aspect of the invention, there is provided the useof a polynucleotide of the invention for therapeutic or prophylacticpurposes, in particular genetic immunization.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet(1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tan, et al. Nature(1992) 356:152, Eisenbraun et at., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seedier et al. PNAS USA(1984) 81: 849).

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells which are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al. MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci, E. coli,streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis,Haemophilus influenzae and Neisseria menigitidis; fungal cells, such ascells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candidaalbictans and Aspergillus; insect cells such as cells of Drosophila S2and Spodoptera St9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK,293, CV-1 and Bowes melanoma cells: and plant cells, such as cells of agymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterolocgous signals.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, ion metalaffinity chromatography (IMAC) is employed for purification. Well knowntechniques for refolding proteins may be employed to regenerate activeconformation when the polypeptide is denatured during intracellularsynthesis, isolation and or purification.

The expression system may also be a recombinant live microorganism, suchas a virus or bacterium. The gene of interest can be inserted into thegenome of a live recombinant virus or bacterium. Inoculation and in vivoinfection with this live vector will lead to in vivo expression of theantigen and induction of immune responses. Viruses and bacteria used forthis purpose are for instance: poxviruses (e.g; vaccinia, fowlpox,canarypox). alphaviruses (Sindbis virus, Semliki Forest Virus,Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associatedvirus, picornaviruses (poliovirus, rhinovirus). herpesviruses (varicellazoster virus, etc), Listeria. Salmonella, Shigella, Neisserin BCG. Theseviruses and bacteria can be virulent, or attenuated in various ways inorder to obtain live vaccines. Such live vaccines also form part of theinvention.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of BASB033 polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof BASB033 polynucleotides and/or polypeptides in a eukaryote,particularly a mammal, and especially a human, will provide a diagnosticmethod for diagnosis of disease, staging of disease or response of aninfectious organism to drugs. Eukaryotes, particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the BASB033 gene or protein, may bedetected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled BASB033 polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence chances at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad Sci. USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingBASB33 nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO:1, 3, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a),

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2, 4 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2, 4.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, 3, which isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan, RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding BASB033 polypeptide can be used to identify andanalyze mutations.

The invention further provides primers with 1, 2, 3 or 4 nucleotidesremoved from the 5′ and/or the 3′ end. These primers may be used for,among other things, amplifying BASB033 DNA and/or RNA isolated from asample derived from an individual, such as a bodily material. Theprimers may be used to amplify a polynucleotide isolated from aninfected individual, such that the polynucleotide may then be subject tovarious techniques for elucidation of the polynucleotide sequence. Inthis way, mutations in the polynucleotide sequence may be detected andused to diagnose and/or prognose the infection or its stage or course,or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing disease,preferably bacterial infections, more preferably infections caused byNeisseria meningitidis, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of SEQ ID NO:1, 3.Increased or decreased expression of a BASB033 polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of BASB033 polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a BASB033 polypeptide, in a sample derived from a host, suchas a bodily material, are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays.Western Blot analysis, antibody sandwich assays, antibody detection andELISA assays.

The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or rids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probe obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularly Neisseriameningitidis, and may be useful in diagnosing and/or prognosing diseaseor a course of disease. A grid comprising a number of variants of thepolynucleotide sequence of SEQ ID NO:1, 3 are preferred. Also preferredis a grid comprising a number of variants of a polynucleotide sequenceencoding the polypeptide sequence of SEQ ID NO:2, 4.

Antibodies

The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

In certain preferred embodiments of the invention there are providedantibodies against BASB033 polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of theinvention can be obtained by administering the polypeptides and/orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975): Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms or animals, such as other mammals, may be usedto express humanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-BASB033 or fromnaive libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks,et al., (199) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al., (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purify the polypeptides or polynucleotides by, for example, affinitychromatography.

Thus, among others, antibodies against BASB033-polypeptide orBASB033-polynucleotide may be employed to treat infections, particularlybacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where the complimentarydetermining region or regions of the hybridoma-derived antibody has beentransplanted into a human monoclonal antibody, for example as describedin Jones et al. (1986), Nature 321, 522-525 or Tempest et al., (1991)Biotechnology 9, 266-273.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverse agonistor inhibitors, in the absence of an agonist or inhibitor, by testingwhether the candidate compound results in inhibition of activation ofthe polypeptide or polynucleotide, as the case may be. Further, thescreenings methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide or polynucleotide ofthe present invention, to form a mixture, measurings BASB033 polypeptideand/or polynucleotide activity in the mixture, and comparing the BASB033polypeptide and/or polynucleotide activity of the mixture to a standard.Fusion proteins, such as those made from Fc portion and BASB033polypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists of thepolypeptide of the present invention, as well as of phylogenetically andand/or functionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleolides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentswhich may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofBASB033 polypeptides or polynucleotides, particularly those compoundsthat are bacteristatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for arsonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising BASB033 polypeptide and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may be a BASB033 agonist or antagonist. Theability of the candidate molecule to agonize or antagonize the BASB033polypeptide is reflected in decreased binding of the labeled ligand ordecreased production of product from such substrate. Molecules that bindgratuitously. i.e., without inducing the effects of BASB033 polypeptideare most likely to be good antagonists. Molecules that bind sell and, asthe case may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocalorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in BASB033 polynucleotide or polypeptideactivity, and binding assays known in the art.

Another example of an assay for BASB033 agonists is a competitive assaythat combines BASB033 and a potential agonist with BASB033-bindingmolecules, recombinant BASB033 binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. BASB033 can be labeled, such as byradioactivity or a colorimetric compound, such that the number ofBASB033 molecules bound to a binding molecule or converted to productcan be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include, among others, small organic molecules,peptides, polypeptides and antibodies that bind to a polynucleotideand/or polypeptide of the invention and thereby inhibit or extinguishits activity or expression. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing BASB033-induced activities,thereby preventing the action or expression of BASB033 polypeptidesand/or polynucleotides by excluding BASB033 polypeptides and/orpolynucleotides from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular bindings molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem, 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of BASB033.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoalobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of anitibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,arsonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial BASB033 proteins that mediate tissue damage and/or; to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

In accordance with yet another aspect of the invention, there areprovided BASB033 agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

In a further aspect, the present invention relates to mimotopes of thepolypeptide of the invention. A mimotope is a peptide sequence,sufficiently similar to the native peptide (sequentially orstructurally), which is capable of being recognised by antibodies whichrecognise the native peptide; or is capable of raising antibodies whichrecognise the native peptide when coupled to a suitable carrier.

Peptide mimotopes may be designed for a particular purpose by addition,deletion or substitution of elected amino acids. Thus, the peptides maybe modified for the purposes of ease of conjugation to a proteincarrier. For example, it may be desirable for some chemical conjugationmethods to include a terminal cysteine. In addition it may be desirablefor peptides conjugated to a protein carrier to include a hydrophobicterminus distal from the conjugated terminus of the peptide, such thatthe free unconjugated end of the peptide remains associated with thesurface of the carrier protein. Thereby presenting the peptide in aconformation which most closely resembles that of the peptide as foundin the context of the whole native molecule. For example, the peptidesmay be altered to have an N-terminal cysteine and a C-terminalhydrophobic amidated tail. Alternatively, the addition or substitutionof a D-stereoisomer form of one or more of the amino acids may beperformed to create a beneficial derivative, for example to enhancestability of the peptide.

Alternatively, peptide mimotopes may be identified using antibodieswhich are capable themselves of binding to the polypeptides of thepresent invention using techniques such as phage display technology (EP0 552 267 B1). This technique, generates a large number, of peptidesequences which mimic the structure of the native peptides and are,therefore, capable of binding to anti-native peptide antibodies, but maynot necessarily themselves share significant sequence homology to thenative polypeptide.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual withBASB033 polynucleotide and/or polypeptide, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Neisseria meningitidis infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector, sequence orribozyme to direct expression of BASB033 polynucleotide and/orpolypeptide, or a fragment or a variant thereof, for expressing BASB033polynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cvtotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example oradministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a BASB033 polynucleotideand/or polypeptide encoded therefrom, wherein the composition comprisesa recombinant BASB033 polynucleotide and/or polypeptide encodedtherefrom and/or comprises DNA and/or RNA which encodes and expresses anantigen of said BASB033 polynucleotide, polypeptide encoded therefrom,or other polypeptide of the invention. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity and/or cellular immunity, such as cellular immunityarising from CTL or CD4+T cells.

A BASB033 polypeptide or a fragment thereof may be fused with co-proteinor chemical moiety which may or may not by itself produce antibodies,but which is capable of stabilizing the first protein and producing afused or modified protein which will have antigenic and/or immunogenicproperties, and preferably protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Haemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, or any other relatively large co-proteinwhich solubilizes the protein and facilitates production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system ofthe organism receiving the protein. The co-protein may be attached toeither the amino- or carboxy-terminus of the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Neisseria meningitidis. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Neisseria meningitidis infection, inmammals, particularly humans.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition o the sterile liquid carrier immediatelyprior to use.

The vaccine formulation of the invention may also include adjuvantsystems for enhancing the immunogenicity of the formulation. Preferablythe adjuvant system raises preferentially a TH1 type of response.

An immune response may be broadly distinguished into two extremecatagories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), and TH2-type immune responses (humoral response).

Extreme TH1-type immune responses may be characterised by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses. In mice TH1-type responses are oftencharacterised by the generation of antibodies of the IgG2a subtype,whilst in the human these correspond to IgG1 type antibodies. TH2-typeimmune responses are characterised by the generation of a broad range ofimmunoglobulin isotypes including in mice IgG1, IgA, and IgM.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

The distinction of TH1 and TH2-type immune responses is not absolute. Inreality an individual will support an immune response which is describedas being predominantly TH1 or predominantly TH2. However, it is oftenconvenient to consider the families of cytokinies in terms of thatdescribed in murine CD4+ve T cell clones by Mosmann and Coffman (Mosmnn,T. R. and Coffman R. L. (1989) TH1 and TH2 cells: different patterns oflymphokine secretion lead to different functional properities, AnnualReview of Immunology, 7, p145-173). Traditionally, TH1-type responsesare associated with the production of the INF-γ and IL-2 cytokines byT-lymphocytes. Other cytokines often directly associated with theinduction of TH1-type immune responses are not produced by T-cells, suchas IL-12. In contrast, TH2-type responses are associated with thesecretion of IL-4, IL-5, IL-6 and IL-13.

It is known that certain vaccine adjuvants are particularly suited tothe stimulation of either TH1 or TH2-type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

Adjuvants which are capable of preferential stimulation of the TH1 cellresponse are described in International Patent Application No. WO94/00153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant.This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains andis manufactured by Ribi Immunochem, Montana. A preferred form of 3De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0689 454 B1 (SmithKline Beecham Biologicals SA).

Preferably, the particles of 3D-MPL are small enough to be sterilefiltered through a 0.22 micron membrane (European Patent number 0 689454). 3D-MPL will be present in the range of 10 μg-100 μg preferably25-50 μg per dose wherein the antigen will typically be present in arange 2-50 μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxicfraction derived from the bark of Quillaja Saponaria Molina. Optionallythis may be admixed with 3 De-O-acylated monophosphoryl lipid A(3D-MPL), optionally together with a carrier.

The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

Combinations of different TH1 stimulating adjuvants, such as thosementioned hereinabove, are also contemplated as providing an adjuvantwhich is a preferential stimulator of TH1 cell response. For example,QS21 can be formulated together with 3D-MPL. The ratio of QS21: 3D-MPLwill typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1and often substantially 1:1. The preferred range for optimal synergy is2.5:1 to 1:1 3D-MPL: QS21.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. In a particularly preferredaspect the antigens in the vaccine composition according to theinvention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg-200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. Preferably the ratio of squalene: alpha tocopherol is equal to orless than 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine compositioncomprising a vaccine formulation of the invention in combination withother antigens, in particular antigens useful for treating, cancers,autoimmune diseases and related conditions. Such a polyvalent vaccinecomposition may include a TH-1 inducing adjuvant as hereinbeforedescribed.

While the invention has been described with reference to certain BASB033polypeptides and polynucleotides, it is to be understood that thiscovers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

The antigen can also be delivered in the form of whole bacteria (dead oralive) or as subcellular fractions, these possibilities do includeN.meningitidis itself.

Compositions, Kits and Administration

In a farther aspect of the invention there are provided compositionscomprising a BASB033 polynucleotide and/or a BASB033 polypeptide foradministration to a cell or to a multicellular organism.

The invention also relates to compositions comprising a polynucleotideand/or a polypeptide discussed herein or their agonists or antagonists.The polypeptides and polynucleotides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a polypeptide and/or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the invention maybe employed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide and/or polynucleotide, such as the soluble form of apolypeptide and/or polynucleotide of the present invention, agonist orantagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other deterrents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable informationresource with which to determine their 2- and 3-dimensional structuresas well as to identify further sequences of similar homology. Theseapproaches are most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as the GCG program package.

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded proteinsequences. Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, DNA, RNA and protein structure analysis, sequence assembly,cladistic analysis, sequence motif analysis, open reading framedetermination, nucleic acid base calling, codon usage analysis, nucleicacid base trimming, and sequencing chromatogram peak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

DEFINITIONS

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M. ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GAP program in the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), and FASTA(Pearson and Lipman Proc. Natl. Acad. Sci. USA85: 244-2448 (1988). The BLAST family of programs is publicly availablefrom NCB1 and other sources (BLAST Manual, Altschul, S., et al., NCB1NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,

Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 8

Gap Length Penalty: 2

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group. MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n _(n) ≦x _(n)−(x _(n) •y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%; 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is: itmay be 100% identical, or it may include up to a certain integer numberof nucleic acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed, either individuallyamong the nucleic acids in the reference sequence or in one or morecontiguous groups within the reference sequence. The number of nucleicacid alterations for a given percent identity is determined bymultiplying the total number of nucleic acids in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleic acids in SEQID NO:1, or:

n _(n) ≦x _(n)−(x _(n) •y),

wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in SEQ ID NO:1, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2). Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to, the referencesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous -groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining, the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) •y).

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.30 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) •y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for themultiplication operator, and wherein any non-integer product or x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Individual(s),” when used herein with reference to an organism, means amulticellular eukaryote, including, but not limited to a metazoan, amammal, an ovid, a bovid, a simian, a primate, and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double-stranded regions.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide chances may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Disease(s)” means any disease caused by or related to infection by abacteria including, for example, upper respiratory tract infection,invasive bacterial diseases, such as bacteremia and meningitis.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Discovery and Confirmatory DNA Sequencing of the BASB033 GeneFrom Two N.meningitidis Strains A: BASB033 in N. meningitidis SerogroupB Strain ATCC13090

The BASB033 gene of SEQ ID NO:1 was first discovered in the IncytePathoSeq database containing unfinished genomic DNA sequences of the N.meningitidis strain ATCC13090. The translation of the BASB033polynucleotide sequence, showed in SEQ ID NO:2, showed significantsimilarity (35% identity in a 292 amino acids overlap) to the Klebsiellapneumoniae outer membrane phospholipase A protein. The sequence of theBASB033 gene was further confirmed experimentally. For this purpose,genomic DNA was extracted from 10¹⁰ cells of the N.meningitidis cells(strain ATCC 13090) using the QIAGEN genomic DNA extraction kit (QiagenGmbh), and 1 μg of this material was submitted to Polymerase ChainReaction DNA amplification using primers Pla-01 (5′-GGT CGA CCA TAT GAATAT ACG GAA TAT GCG CTA-3′) [SEQ ID NO:5] containing an internal NdeIsite (underlined) and Pla-02 (5′-CGC CGC TCG AGG ATG CCG TCC AAG TCGTTG-3′) [SEQ ID NO:6] containing an internal XhoI site (underlined).This PCR product was gel-purified and subjected to DNA sequencing usingthe Big Dye Cycle Sequencing kit (Perkin-Elmer) and an ABI 373A/PRISMDNA sequencer. DNA sequencing was performed on both strands with aredundancy of 2 and the full-length sequence was assembled using theSeqMan program from the DNASTAR Lasergene software package. Theresulting DNA sequence turned out to be 100% identical to SEQ ID NO:1.

B: BASB033 in N. meningitidis Serogroup B Strain H44/76

The sequence of the BASB033 gene was also determined in another N.meningitidis serogroup B strain, the strain H44/76. For this purpose,genomic DNA was extracted from the N. meningitidis strain H44/76 usingthe experimental conditions presented in Example 1. This material (1 μg)was then submitted to Polymerase Chain Reaction DNA amplification usingprimers Pla-01 and Pla-02 specific for the BASB033 gene. A ˜1100 bp DNAfragment was obtained, digested by the NdeI/XhoI restrictionendonucleases and inserted into the corresponding sites of the pET-24bcloning/expression vector (Novagen) using standard molecular biologytechniques (Molecular Cloning, a Laboratory Manual, Second Edition, Eds:Sambrook. Fritsch & Maniatis, Cold Spring Harbor press 1989).Recombinant pET-24b/BASB033 was then submitted to DNA sequencing usingthe Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNAsequencer in the conditions described by the supplier. As a result, thepolynucleotide and deduced polypeptide sequences, referred to as SEQ IDNO:3 and SEQ ID NO:4 respectively, were obtained. Using the MegAlignprogram in the DNASTAR Lasergene package, an alignment of thepolynucleotide sequences of SEQ ID NO:1 and 3 was performed, and isdisplayed in FIG. 1; their level of identity amounts to 99.3%, asdetermined by the program. Using the same MegAlign program, an alignmentof the polypeptide sequences of SEQ ID NO:2 and 4 was performed, and isdisplayed in FIG. 2; their level of identity amounts to 98.9%, asdetermined by the program.

Taken together, these data indicate strong sequence conservation of theBASB033 gene among the two N.meningitidis serogroup B strains.

Example 2 Expression and Purification of Recombinant BASB033 Protein inEscherichia coli

The construction of the pET24b/BASB033 cloning/expression vector wasdescribed in Example 1B. This vector harbours the BASB033 gene isolatedfrom the strain H44/76 in fusion with a stretch of 6 Histidine residues,placed under the control of the strong bacteriophage T7 gene 10promoter. For expression study, this vector was introduced into theEscherichia coli strain Novablue (DE3) (Novagen), in which, the gene forthe T7 polymerase is placed under the control of the isopropyl-beta-Dthiogalactoside (IPTG)-regulatable lac promoter. Liquid cultures (100ml) of the Novablue (DE3) [pET-24b/BASB033)] E. coli recombinant strainwere grown at 37° C. under agitation until the optical density at 600 nm(0D600) reached 0.6. At that time-point, IPTG was added at a finalconcentration of 1 mM and the culture was grown for 4 additional hours.The culture was then centrifuged at 10,000 rpm and the pellet was frozenat −20° C. for at least 10 hours.

After thawing, the pellet (3 liter culture) was resuspended in 20 mMphosphate buffer pH 8.0 containing 20 units benzonase per ml andincubated at 22° C. for 30 min. Lysed cells were pelleted 30 min at15,000 rpm (Beckman J2-HS centrifuge, JA-20 rotor) at 4° C. Therecombinant protein BASB033/His6 was solubilised by 8 M Urea, 20 mMphosphate pH 8.0 overnight at 4° C. Cell debris were pelleted 30 min at15,000 rpm in a JA-20 rotor at 4° C. The sample was loaded at aflow-rate of 1 m/min on a 4 ml Fractogel EMD S03⁻ 650S column (Merck).The column was equilibrated in 8 M Urea, 20 mM phosphate pH 8.0. Afterpassage of the flowthrough, the column was washed with equilibrationbuffer until the base line was reached. The recombinant protein waseluted from the column by 100 mM NaCl in 8M Urea, 20 mM phosphate pH8.0, at 1 ml/min. Eluted sample was dialysed at 4° C. versus PBScontaining 0.5 M Arginine. As shown in FIG. 3 (lane 6), an enriched(purity estimated at 50% pure in CBB stained SDS-PAGE) BASB033/His6protein, migrating at 43 kDa (estimated relative molecular mass), waseluted from the column. This polypeptide was reactive against a mousemonoclonal antibody raised against the 5-histidine motif (see FIG. 4,lane 6). Taken together, these data indicate that the BASB033 gene canbe expressed and purified under a recombinant form (BASB033/His6) in E.coli.

Example 3 Immunization of Mice With Recombinant BASB033

Partially purified recombinant BASB033 expressed in E. coli has beeninjected three times in Balb/C mice on days 0. 14 and 28 (10animals/group). Animals were injected by the subcutaneous route with 5μg of antigen adsorbed on 100 μg AlPO₄. A negative control groupconsisting of mice immunized with the SBAS2 adjuvant only has also beenadded in the experiment. Mice were bled on days 28 (14 days Post II) and35 (7 days Post III) in order to detect specific anti-BASB033antibodies. Specific anti-BASB033 antibodies were measured by Elisa onpooled sera on the recombinant BASB033 protein as well as on E. coliproteins. Anti-BASB033 response has also been evaluated bywestern-blotting using the recombinant antigen.

Elisa results with coated BASB033 are presented hereafter, and show thatBASB033 antigen is clearly immunogenic in mice, although weakly, anddespite its partial purity (FIG. 5). The difference observed between thespecific BASB033 and E. coli response is due to specific anti-BASB033antibodies. There is no specific BASB033 response in mice from thenegative control group. This is also clearly demontrated in FIG. 6, inwhich there is clear BASB033 band detected at around 43 kD. Sera fromPost II or Post III bleeding were used for these assays.

Recognition of the BASB033 epitopes on different NmB strains bywestern-blotting In this test, immunized mice sera (pooled) have beentested by western-blotting for recognition of the BASB033 epitopes onsix different Neisseria meningitidis B strains: H44/76 (B:15:P1.7, 16,lineage ET-5), M97 250687 (B:4:P1.15), BZ10 (B:2b:P1.2, lineage A4),BZ198 (B:NT*: -, lineage 3), and EG328 (B:NT*, lineage ST-18), and onpartially purified recombinant BASB033 protein. (*: NT: Not Typed).Briefly, 15 μl (>10⁸ cells/lane) of each sample treated with samplebuffer (10 min at 95° C.) are put into a SDS-PAGE gradient gel(Tris-glycine 4-20%. Novex, code n°EC6028). Electrophoretic migrationoccurs at 35 mA/gel for 90 min. Afterwards, proteins are transferred tonitrocellulose sheet (0.45 μm, Bio-rad code n° 162-0114) at 100 voltsfor 1 hour using a Bio-rad Trans-blot system (code n°170-3930). Filterwas blocked with PBS-0.05% Tween 20 overnight at room temperature,before incubation with the mice sera containing the anti-BASB033antibodies. These sera are diluted 100 times in PBS-0.05% Tween 20, andincubated on the nitrocellulose sheet for two hours at room temperaturewith gentle shaking, using a mini-blotter system (Miniprotean, Bio-radcode n° 170-4017). After three repeated washing steps in PBS-0.05% Tween20 for 5 min., the nitrocellulose sheet is incubated at room temperaturefor 1 hour under gentle shaking with the appropriate conjugate(biotinylated anti-mouse 1 g antibodies from sheep. Amersham coden°RPN1001) diluted at 1/500 in the same washing buffer. The membrane iswashed three times as previously, and incubated for 30 min withagitation using the streptavidin-peroxidase complex (Amersham coden°1051) diluted at 1/1000 in the washing buffer. After the last threerepeated washing steps, the revelation occurs during the 20 minincubation time in a 50 ml solution containing 30 mg 4-chloro-1-naphtol(Sigma), 10 ml methanol, 40 ml PBS, and 30 μl of H₂O₂. The staining isstopped while washing the membrane several times in distillated water.

Results illustrated hereafter in FIGS. 7 and 8 show that almost allstrains tested present a band around 43 kD, meaning that antibodiesdirected against the recombinant BASB033 protein recognize the nativeprotein at the surface of Neisseria meningitidzs B cells (6/7 strainsare recognized in this case). Then, BASB033 protein is probablyexpressed in a majority of the N.meningitidis B strains. All other bandscould be due to antibodies directed against BASB033 aggregation products(as the one observed around 95-100 kD), related products, orcross-reacting antigens between E. coli and Neiseria meningitidis Bbacteria, since the preparation used for immunization still containedE.coli contaminants. There is no reaction band observed at 43 kD on E.coli proteins, meaning that the antigen is not present in E.coli.

Example 4 Presence of Anti-BASB033 Antibodies in Sera from ConvalescentPatients

In this test, a few convalescent sera have been tested bywestern-blotting for recognition of the purified recombinant BASB033protein. Briefly, 24 μg of partially purified BASB033 protein are putinto a SDS-PAGE gradient gel (4-20%, Novex, code n°EC6029) forelectrophoretic migration. Proteins are transferred to nitrocellulosesheet (0.45 μm, Bio-rad code n° 162-0114) at 100 volts for 1 hour usinga Bio-rad Trans-blot system (code n°170-3930). Afterwards, filter isblocked with PBS-0.05% Tween 20 overnight at room temperature, beforeincubation with the human sera. These sera are diluted at 1/50 inPBS-0.05% Tween 20, and incubated on the nitrocellulose sheet for twohours at room temperature with gentle shaking, using a mini-blottersystem (Miniprotean, Bio-rad code n° 170-4017). After three repeatedwashing steps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheetis incubated at room temperature for 1 hour under gentle shaking withthe appropriate conjugate (biotinylated anti-human Ig antibodies, fromsheep, Amersham code n°RPN1003) diluted at 1/500 in the same washingbuffer. The membrane is washed three times as previously, and incubatedfor 30 min with agitation using the streptavidin-peroxidase complex(Amersham code n°1051) diluted at 1/1000 in the washing buffer. Afterthe last three repeated washing steps, the revelation occurs during the20 min incubation time in a 50 ml solution containing 30 mg4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml of PBS, and 30 μl ofH₂O₂. The staining is stopped while washing the membrane several timesin distillated water.

Results illustrated in FIG. 9 (Part B) show that all the 7 convalescentsera tested react against the BASB033 recombinant protein at around 43kD, as the BASB033 band is clearly visible. The weakest response isobserved with the 260601 convalescent serum. This response supports thepotential use of BASB033 antigen as vaccine component. In part A of thewestern-blot, we confirm that mice sera recognize very well the intactrecombinant BASB033 protein as previously discussed.

Example 5 Analysis of the Non-coding Flanking Regions of the BASB033Gene, and its Exploitation for Modulated BASB033 Gene Expression

The non-coding flanking regions of the BASB033 gene contain regulatoryelements important in the expression of the gene. This regulation takesplace both at the transcriptional and translational level. The sequenceof these regions, either upstream or downstream of the open readingframe of the gene; can be obtained by DNA sequencing. This sequenceinformation allows the determination of potential regulatory motifs suchas the different promoter elements, terminator sequences, induciblesequence elements, repressors, elements responsible for phase variation,the shine-dalgarno sequence, regions with potential secondary structureinvolved in regulation, as well as other types of regulatory motifs orsequences.

This sequence information allows the modulation of the naturalexpression of gene BASB033. The upregulation of the gene expression maybe accomplished by altering the promoter, the shine-dalgarno sequence,potential repressor or operator elements, or any other elementsinvolved. Likewise, downregulation of expression can be achieved bysimilar types of modifications. Alternatively, by changing phasevariation sequences, the expression of the gene can be put under phasevariation control, or may be uncoupled from this regulation. In anotherapproach, the expression of the gene can be put under the control of oneor more inducible elements allowing regulated expression. Examples ofsuch regulation include, but are not limited to, induction bytemperature shift, addition of inductor substrates like selectedcarbohydrates or their derivatives, trace elements, vitamins,co-factors, metal ions, etc.

Such modifications as described above can be introduced by severaldifferent means. The modification of sequences involved in geneexpression can be done in vivo by random mutagenesis followed byselection for the desired phenotype. Another approach consists inisolating the region of interest and modifying it by random mutagenesis,or site-directed mutagenesis, insertion or deletion mutagenesis. Themodified region can then be reintroduced into the bacterial genome byhomologous recombination, and the effect on gene expression can beassessed. In another approach, the sequence knowledge of the region ofinterest can be used to replace or delete all or part of the naturalregulatory sequences. In this case, the regulatory region targeted isisolated and modified so as to contain the regulatory elements fromanother gene, a combination of regulatory elements from different genes,a synthetic regulatory region, or any other regulatory region, or todelete selected parts of the wild-type regulatory sequences. Thesemodified sequences can then be reintroduced into the bacterium viahomologous recombination into the genome. A non-exhaustive list ofpreferred promoters that could be used for up-regulation of geneexpression includes the promoter porA. por, lbpB. tbpB, p110, 1 st,hpuAB from N. meningitidis or N. gonorroheae.

In one example, the expression of the gene can be modulated byexchanging its promoter with a stronger promoter (through isolating theupstream sequence of the gene, in vitro modification of this sequence,and reintroduction into the genome by homologous recombination).Upregulated expression can be obtained in both the bacterium as well asin the outer membrane vesicles shed (or made) from the bacterium. Inother examples, the described approaches can be used to generaterecombinant bacterial strains with improved characteristics for vaccineapplications. These can be, but are not limited to, attenuated strains,strains with increased expression of selected antigens, strains withknockouts (or decreased expression) of genes interfering with the immuneresponse, strains with modulated expression of immunodominant proteins,strains with modulated shedding of outer-membrane vesicles.

A region directly upstream of the BASB033 gene is given in the sequenceof SEQ ID NO:7. This sequence is a further aspect of the invention.

FIGURE LEGENDS

FIG. 3:

A substantially purified (estimated at 50%) BASB033 protein fraction wasseparated on a 4-20% gradient polyacrylamide gel (NOVEX) under SDS-PAGEconditions in parallel to a protein molecular weight marker (lane 1),then stained with Coomassie blue. Lane 6 clearly appears enriched withBASB033 at around 43 kD (lanes 2 and 3 are total cellular proteinextract, lane 4 is the flowthrough, lanes 5 to 10 are the elutionprofile).

FIG. 4:

A substantially purified (estimated at 50%) BASB033 protein fraction wasseparated on a 4-20% gradient polyacrylamide gel (NOVEX) under SDS-PAGEconditions in parallel to a protein molecular weight marker (lane 1),then analyzed by western blot using an anti-His5 mouse monoclonalantibody. Lane 6 clearly reveals the BASB033 polypeptide at around 43 kD(lanes 2 and 3 are total cellular protein extract, lane 4 is theflowthrough, lanes 5 to 10 are the elution profile).

BASB033 Polynucleotide and Polypeptide Sequences

SEQ ID NO:1

Neisseria meningitidis BASB033 polynucleotide sequence

ATGAATATACGGAATATGCGCTATATCCTTTTGACAGGACTGTTGCCGACGGCATCCGCTTTTGGAGAGACCGCGCTGCAATGCGCCGCTTTGACGGACAATGTTACGCGTTTGGTGTGTTACGACAGGATTTTTGCGGCACAGCTTCCGTCTTCGGCAGGGCAGGAAGGGCAGGAGTCGAAAGCCGTACTCAATCTGACGGAAACCGTCCGCAGCAGCCTGGATAAGGGCGAGGCGGTCATTGTTGTTGAAAAAGGCGGGGATGCGCTTCCTGCCGACAGTGCGGGCGAAACCGCCGACATCTATACGCCTTTGAGCCTGATGTACGACTTGGACAAAAACGATTTGCGCGGGCTGTTGGGCGTACGCGAACACAATCCGATGTACCTTATGCCGCTCTGGTACAACAATTCGCCCAACTATGCCCCGAGTTCGCCGACGCGCGGTACAACTGTACAGGAAAAATTCGGACAGCAGAAACGTGCGGAAACCAAATTGCAGGTTTCGTTCAAAAGCAAAATTGCCGAAGATTTGTTTAAAACCCGCGCGGATCTGTGGTTCGGCTACACCCAAAGATCCGATTGGCAGATTTACAACCAAGGCAGGAAATCCGCGCCGTTCCGCAATACGGATTACAAACCTGAAATTTTCCTGACCCAGCCTGTGAAGGCGGATTTGCCGTTCGGCGGCAGGCTGCGTATGCTCGGTGCGGGTTTTGTCCACCAGTCCAACGGACAGAGCCGTCCCGAATCGCGTTCGTGGAACAGGATTTACGCCATGGCAGGCATGGAATGGGGCAAATTGACGGTGATTCCGCGCGTGTGGGTGCGTGCGTTCGATCAGAGCGGCGATAAAAACGACAATCCCGATATTGCCGACTATATGGGGTATGGCGACGTGAAGCTGCAGTACCGCCTGAACGACAGGCAGAATGTGTATTCCGTATTGCGCTACAACCCCAAAACGGGCTACGGCGCGATTGAAGCCGCCTACACGTTTCCGATTAAGGGCAAACTCAAAGGCGTGGTACGCGGATTCCACGGTTACGGCGAGAGCCTGATCGACTACAACCACAAGCAGAACGGTATCGGTATCGGGTTGATGTTCAACGACTTGGACGGCATCTGA

SEQ ID NO:2

Neisseria meningitidis BASB033 polypeptide sequence deduced from thepolynucleotide sequence of SEQ ID NO:1

MNIRNMRYILLTGLLPTASAFGETALQCAALTDNVTRLVCYDRIFAAQLPSSAGQEGQESKAVLNLTETVRSSLDKGEAVIVVEKGGDALPADSAGETADIYTPLSLMYDLDKNDLRGLLGVREHNPMYLMPLWYNNSPNYAPSSPTRGTTVQEKFGQQKRAETKLQVSFKSKIAEDLFKTRADLWFGYTQRSDWQIYNQGRKSAPFRNTDYKPEIFLTQPVKADLPFGGRLRMLGAGFVHQSNGQSRPESRSWNRIYAMAGMEWGKLTVIPRVWVRAFDQSGDKNDNPDIADYMGYGDVKLQYRLNDRQNVYSVLRYNPKTGYGAIEAAYTFPIKGKLKGVVRGFHGYGESLIDYNHKQNGIGIGLMFNDLDGI

SEQ ID NO:3

Neisseria menigitidis BASB033 polynucleotide sequence from strain H44/76

ATGAATATACGGAATCGCTATATTCTTTTGACAGGACTGTTGCCGATGGCATCCGCTTTTGGAGAGACCGCGCTGCAATGCGCCGCTTTGACGGACAATGTTACGCGTTTGGCGTGTTACGACAGGATTTTTGCGGCACAGCTTCCGTCTTCGGCAGGGCAGGAAGGGCAGGAGTCGAAAGCCGTACTCAATCTGACGGAAACCGTCCGCAGCAGCCTGGATAAGGGCGAGGCGGTCATTGTTGTTGAAAAAGGCGGGGATGCGCTTCCTGCCGACAGTGCGGGCGAAACCGCCGACATCTATACGCCTTTGAGCCTGATGTACGACTTGGACAAAAACGATTTGCGCGGGCTGTTGGGCGTACGCGAACACAATCCGATGTACCTTATGCCGCTCTGGTACAACAATTCGCCCAACTATGCCCCGgGTTCGCCGACGCGCGGTACgACTGTACAGGAAAAATTCGGACAGCACAAACGTGCGGAAACCAAATTGCAGGTTTCGTTCAAAAGCAAAATTGCCGAAGATTTGTTTAAAACCCGCGCGGATCTGTGGTTCGGCTACACCCAAAGATCCGATTGGCAGATTTACAACCAAGGCAGGAAATCCGCGCCGTTCCGCAATACGGATTACAAACCTGAAATTTTCCTGACCCAGCCTGTGAAGGCGGATTTGCCGTTCGGCGGCAGGCTGCGTATGCTCGGTGCGGGTTTTGTCCACCAGTCCAACGGACAGAGCCGTCCCGAATCGCGTTCGTGGAACAGGATTTACGCCATGGCAGGCATGGAATGGGGCAAATTGACGGTGATTCCGCGCGTGTGGGTGCGTGCGTTCGATCAGAGCGGCGATAAAAACGACAATCCCGATATTGCCGACTATATGGGGTATGGCGACGTGAAGCTGCAGTACCGCCTGAACGACAGGCAGAATGTGTATTCCGTATTGCGCTACAACCCCAAAACGGGCTACGGCGCGATTGAAGCCGCCTACACGTTTCCGATTAAGGGCAAACTCAAAGGCGTGGTACGCGGATTCCACGGTTACGGCGAGAGCCTGATCGACTACAACCACAAGCAGAACGGTATCGGTATCGGGTTGATGTTCAACGACTTGGACGGCATCTGA

SEQ ID NO:4

Neisseria meningitidis BASB033 polypeptide sequence deduced from thepolynucleotide sequence of SEQ ID NO:3

MNIRNRYILLTGLLPMASAFGETALQCAALTDNVTRLACYDRIFAAQLPSSAGQEGQESKAVLNLTETVRSSLDKGEAVIVVEKGGDALPADSAGETADIYTPLSLMYDLDKNDLRGLLGVREHNPMYLMPLWYNNSPNYAPGSPTRGTTVQEKFGQQKRAETKLQVSFKSKIAEDLFKTRADLWFGYTQRSDWQIYNQGRKSAPFRNTDYKPEIFLTQPVKADLPFGGRLRMLGAGFVHQSNGQSRPESRSWNRIYAMAGMEWGKLTVIPRVWVRAFDQSGDKNDNPDIADYMGYGDVKLQYRLNDRQNVYSVLRYNPKTGYGAIEAAYTFPIKGKLKGVVRGFHGYGESLIDYNHKQNGIGIGLMFNDLDGI

SEQ ID NO:5

GGT CGA CCA TAT GAA TAT ACG GAA TAT GCG CTA

SEQ ID NO:6

CGC CGC TCG AGG ATG CCG TCC AAG TCG TTG

SEQ ID NO:7

CGTACCGCATTCCGCACTGCAGTGAAAAAAGTATTGAAAGCAGTCGAAGCAGGCGATAAAGCTGCCGCACAAGCGGTTTACCAAGAGTCCGTCAAAGTCATCGACCGCATCGCCGACAAGGGCGTGTTCCATAAAAACAAAGCGGCTCGCCACAAAACCCGTTTGTCTCAAAAAGTAAAACCTTGGCTTGATTTTTGCAAAACCTGCAATCCGGTTTTCATCGTCGATTCCGAAAACCCCTGAAGCCCGACGGTTTCGGGGTTTTCTGTATTGCGGGGACAAAATCCCGAAATGGCGGAAAGGGTGCGGTTTTTTATCCGAATCCGCTATAAAATGCCGTCTGAAAACCAATATGCCGACAATGGGGGTGGAG

Deposited Materials

A deposit containing a Neisseria meningitidis Serogroup B strain hasbeen deposited with the American Type Culture Collection (herein “ATCC”)on Jun. 22, 1997 and assigned deposit number 13090. The deposit wasdescribed as Neisseria meningitidis (Albrecht and Ghon) and is afreeze-dried, 1.5-2.9 kb insert library constructed from N. meningitidisisolate. The deposit is described in Int. Bull. Bacteriol. Nomencl.Taxon. 8: 1-15 (1958).

The Neisseria meningitidis strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains the full length BASB033 gene. The sequenceof the polynucleotides contained in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

7 1 1128 DNA Neisseria meningitidis 1 atgaatatac ggaatatgcg ctatatccttttgacaggac tgttgccgac ggcatccgct 60 tttggagaga ccgcgctgca atgcgccgctttgacggaca atgttacgcg tttggtgtgt 120 tacgacagga tttttgcggc acagcttccgtcttcggcag ggcaggaagg gcaggagtcg 180 aaagccgtac tcaatctgac ggaaaccgtccgcagcagcc tggataaggg cgaggcggtc 240 attgttgttg aaaaaggcgg ggatgcgcttcctgccgaca gtgcgggcga aaccgccgac 300 atctatacgc ctttgagcct gatgtacgacttggacaaaa acgatttgcg cgggctgttg 360 ggcgtacgcg aacacaatcc gatgtaccttatgccgctct ggtacaacaa ttcgcccaac 420 tatgccccga gttcgccgac gcgcggtacaactgtacagg aaaaattcgg acagcagaaa 480 cgtgcggaaa ccaaattgca ggtttcgttcaaaagcaaaa ttgccgaaga tttgtttaaa 540 acccgcgcgg atctgtggtt cggctacacccaaagatccg attggcagat ttacaaccaa 600 ggcaggaaat ccgcgccgtt ccgcaatacggattacaaac ctgaaatttt cctgacccag 660 cctgtgaagg cggatttgcc gttcggcggcaggctgcgta tgctcggtgc gggttttgtc 720 caccagtcca acggacagag ccgtcccgaatcgcgttcgt ggaacaggat ttacgccatg 780 gcaggcatgg aatggggcaa attgacggtgattccgcgcg tgtgggtgcg tgcgttcgat 840 cagagcggcg ataaaaacga caatcccgatattgccgact atatggggta tggcgacgtg 900 aagctgcagt accgcctgaa cgacaggcagaatgtgtatt ccgtattgcg ctacaacccc 960 aaaacgggct acggcgcgat tgaagccgcctacacgtttc cgattaaggg caaactcaaa 1020 ggcgtggtac gcggattcca cggttacggcgagagcctga tcgactacaa ccacaagcag 1080 aacggtatcg gtatcgggtt gatgttcaacgacttggacg gcatctga 1128 2 375 PRT Neisseria meningitidis 2 Met Asn IleArg Asn Met Arg Tyr Ile Leu Leu Thr Gly Leu Leu Pro 1 5 10 15 Thr AlaSer Ala Phe Gly Glu Thr Ala Leu Gln Cys Ala Ala Leu Thr 20 25 30 Asp AsnVal Thr Arg Leu Val Cys Tyr Asp Arg Ile Phe Ala Ala Gln 35 40 45 Leu ProSer Ser Ala Gly Gln Glu Gly Gln Glu Ser Lys Ala Val Leu 50 55 60 Asn LeuThr Glu Thr Val Arg Ser Ser Leu Asp Lys Gly Glu Ala Val 65 70 75 80 IleVal Val Glu Lys Gly Gly Asp Ala Leu Pro Ala Asp Ser Ala Gly 85 90 95 GluThr Ala Asp Ile Tyr Thr Pro Leu Ser Leu Met Tyr Asp Leu Asp 100 105 110Lys Asn Asp Leu Arg Gly Leu Leu Gly Val Arg Glu His Asn Pro Met 115 120125 Tyr Leu Met Pro Leu Trp Tyr Asn Asn Ser Pro Asn Tyr Ala Pro Ser 130135 140 Ser Pro Thr Arg Gly Thr Thr Val Gln Glu Lys Phe Gly Gln Gln Lys145 150 155 160 Arg Ala Glu Thr Lys Leu Gln Val Ser Phe Lys Ser Lys IleAla Glu 165 170 175 Asp Leu Phe Lys Thr Arg Ala Asp Leu Trp Phe Gly TyrThr Gln Arg 180 185 190 Ser Asp Trp Gln Ile Tyr Asn Gln Gly Arg Lys SerAla Pro Phe Arg 195 200 205 Asn Thr Asp Tyr Lys Pro Glu Ile Phe Leu ThrGln Pro Val Lys Ala 210 215 220 Asp Leu Pro Phe Gly Gly Arg Leu Arg MetLeu Gly Ala Gly Phe Val 225 230 235 240 His Gln Ser Asn Gly Gln Ser ArgPro Glu Ser Arg Ser Trp Asn Arg 245 250 255 Ile Tyr Ala Met Ala Gly MetGlu Trp Gly Lys Leu Thr Val Ile Pro 260 265 270 Arg Val Trp Val Arg AlaPhe Asp Gln Ser Gly Asp Lys Asn Asp Asn 275 280 285 Pro Asp Ile Ala AspTyr Met Gly Tyr Gly Asp Val Lys Leu Gln Tyr 290 295 300 Arg Leu Asn AspArg Gln Asn Val Tyr Ser Val Leu Arg Tyr Asn Pro 305 310 315 320 Lys ThrGly Tyr Gly Ala Ile Glu Ala Ala Tyr Thr Phe Pro Ile Lys 325 330 335 GlyLys Leu Lys Gly Val Val Arg Gly Phe His Gly Tyr Gly Glu Ser 340 345 350Leu Ile Asp Tyr Asn His Lys Gln Asn Gly Ile Gly Ile Gly Leu Met 355 360365 Phe Asn Asp Leu Asp Gly Ile 370 375 3 1125 DNA Neisseriameningitidis 3 atgaatatac ggaatcgcta tattcttttg acaggactgt tgccgatggcatccgctttt 60 ggagagaccg cgctgcaatg cgccgctttg acggacaatg ttacgcgtttggcgtgttac 120 gacaggattt ttgcggcaca gcttccgtct tcggcagggc aggaagggcaggagtcgaaa 180 gccgtactca atctgacgga aaccgtccgc agcagcctgg ataagggcgaggcggtcatt 240 gttgttgaaa aaggcgggga tgcgcttcct gccgacagtg cgggcgaaaccgccgacatc 300 tatacgcctt tgagcctgat gtacgacttg gacaaaaacg atttgcgcgggctgttgggc 360 gtacgcgaac acaatccgat gtaccttatg ccgctctggt acaacaattcgcccaactat 420 gccccgggtt cgccgacgcg cggtacgact gtacaggaaa aattcggacagcagaaacgt 480 gcggaaacca aattgcaggt ttcgttcaaa agcaaaattg ccgaagatttgtttaaaacc 540 cgcgcggatc tgtggttcgg ctacacccaa agatccgatt ggcagatttacaaccaaggc 600 aggaaatccg cgccgttccg caatacggat tacaaacctg aaattttcctgacccagcct 660 gtgaaggcgg atttgccgtt cggcggcagg ctgcgtatgc tcggtgcgggttttgtccac 720 cagtccaacg gacagagccg tcccgaatcg cgttcgtgga acaggatttacgccatggca 780 ggcatggaat ggggcaaatt gacggtgatt ccgcgcgtgt gggtgcgtgcgttcgatcag 840 agcggcgata aaaacgacaa tcccgatatt gccgactata tggggtatggcgacgtgaag 900 ctgcagtacc gcctgaacga caggcagaat gtgtattccg tattgcgctacaaccccaaa 960 acgggctacg gcgcgattga agccgcctac acgtttccga ttaagggcaaactcaaaggc 1020 gtggtacgcg gattccacgg ttacggcgag agcctgatcg actacaaccacaagcagaac 1080 ggtatcggta tcgggttgat gttcaacgac ttggacggca tctga 1125 4374 PRT Neisseria meningitidis 4 Met Asn Ile Arg Asn Arg Tyr Ile Leu LeuThr Gly Leu Leu Pro Met 1 5 10 15 Ala Ser Ala Phe Gly Glu Thr Ala LeuGln Cys Ala Ala Leu Thr Asp 20 25 30 Asn Val Thr Arg Leu Ala Cys Tyr AspArg Ile Phe Ala Ala Gln Leu 35 40 45 Pro Ser Ser Ala Gly Gln Glu Gly GlnGlu Ser Lys Ala Val Leu Asn 50 55 60 Leu Thr Glu Thr Val Arg Ser Ser LeuAsp Lys Gly Glu Ala Val Ile 65 70 75 80 Val Val Glu Lys Gly Gly Asp AlaLeu Pro Ala Asp Ser Ala Gly Glu 85 90 95 Thr Ala Asp Ile Tyr Thr Pro LeuSer Leu Met Tyr Asp Leu Asp Lys 100 105 110 Asn Asp Leu Arg Gly Leu LeuGly Val Arg Glu His Asn Pro Met Tyr 115 120 125 Leu Met Pro Leu Trp TyrAsn Asn Ser Pro Asn Tyr Ala Pro Gly Ser 130 135 140 Pro Thr Arg Gly ThrThr Val Gln Glu Lys Phe Gly Gln Gln Lys Arg 145 150 155 160 Ala Glu ThrLys Leu Gln Val Ser Phe Lys Ser Lys Ile Ala Glu Asp 165 170 175 Leu PheLys Thr Arg Ala Asp Leu Trp Phe Gly Tyr Thr Gln Arg Ser 180 185 190 AspTrp Gln Ile Tyr Asn Gln Gly Arg Lys Ser Ala Pro Phe Arg Asn 195 200 205Thr Asp Tyr Lys Pro Glu Ile Phe Leu Thr Gln Pro Val Lys Ala Asp 210 215220 Leu Pro Phe Gly Gly Arg Leu Arg Met Leu Gly Ala Gly Phe Val His 225230 235 240 Gln Ser Asn Gly Gln Ser Arg Pro Glu Ser Arg Ser Trp Asn ArgIle 245 250 255 Tyr Ala Met Ala Gly Met Glu Trp Gly Lys Leu Thr Val IlePro Arg 260 265 270 Val Trp Val Arg Ala Phe Asp Gln Ser Gly Asp Lys AsnAsp Asn Pro 275 280 285 Asp Ile Ala Asp Tyr Met Gly Tyr Gly Asp Val LysLeu Gln Tyr Arg 290 295 300 Leu Asn Asp Arg Gln Asn Val Tyr Ser Val LeuArg Tyr Asn Pro Lys 305 310 315 320 Thr Gly Tyr Gly Ala Ile Glu Ala AlaTyr Thr Phe Pro Ile Lys Gly 325 330 335 Lys Leu Lys Gly Val Val Arg GlyPhe His Gly Tyr Gly Glu Ser Leu 340 345 350 Ile Asp Tyr Asn His Lys GlnAsn Gly Ile Gly Ile Gly Leu Met Phe 355 360 365 Asn Asp Leu Asp Gly Ile370 5 33 DNA Artificial Sequence Primer Sequence 5 ggtcgaccat atgaatatacggaatatgcg cta 33 6 30 DNA Artificial Sequence Primer Sequence 6cgccgctcga ggatgccgtc caagtcgttg 30 7 373 DNA Neisseria meningitidis 7cgtaccgcat tccgcactgc agtgaaaaaa gtattgaaag cagtcgaagc aggcgataaa 60gctgccgcac aagcggttta ccaagagtcc gtcaaagtca tcgaccgcat cgccgacaag 120ggcgtgttcc ataaaaacaa agcggctcgc cacaaaaccc gtttgtctca aaaagtaaaa 180ccttggcttg atttttgcaa aacctgcaat ccggttttca tcgtcgattc cgaaaacccc 240tgaagcccga cggtttcggg gttttctgta ttgcggggac aaaatcccga aatggcggaa 300agggtgcggt tttttatccg aatccgctat aaaatgccgt ctgaaaacca atatgccgac 360aatgggggtg gag 373

What is claimed is:
 1. An isolated polypeptide comprising a member selected from the group consisting of (a) an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4; (b) an immunogenic polypeptide comprising a fragment sequence of at least 15 amino acids that corresponds to an aligned contiguous segment of SEQ ID NO:2 or SEQ ID NO:4; wherein the isolated polypeptide, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell immune response to a polypeptide having the sequence of SEQ ID NO:2 or SEQ ID NO:4.
 2. The isolated polypeptide of claim 1, wherein the polypeptide is the polypeptide of (a).
 3. The isolated polypeptide of claim 1, wherein the polypeptide is the polypeptide of (b).
 4. The isolated polypeptide of claim 1, wherein the immunogenic fragment of (b) comprises at least 20 amino acids.
 5. The isolated polypeptide of claim 1, wherein the isolated polypeptide consists of SEQ ID NO:2 or SEQ ID NO:4.
 6. A fusion protein comprising the isolated polypeptide of claim
 1. 7. An immunogenic composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
 8. The immunogenic composition of claim 7, wherein the vaccine comprises at least one other Neisseria meningitidis antigen. 